Generation of porcine fetal fibroblasts expressing the tetracycline-inducible Cas9 gene by somatic cell nuclear transfer.
Generation of porcine fetal fibroblasts expressing the tetracycline-inducible Cas9 gene by somatic cell nuclear transfer.
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通过体细胞核移植产生表达四环素诱导的Cas9基因的猪胎儿成纤维细胞
DOI：
10.3892/mmr.2016.5530
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发表时间：
2016-09
影响因子：
3.4
通讯作者：
Zhang S
中科院分区：
文献类型：
--
作者：
Liu G;Liu K;Wei H;Li L;Zhang S
Cas9 核酸内切酶来自化脓性链球菌所谓的成簇规则间隔短回文重复序列 (CRISPR)/CRISPR 相关 (Cas) 系统，II 型作为 RNA 引导的核酸内切酶，可编辑原核和真核生物的基因组，包括通过 DNA 双链断裂修复机制进行删除和插入。在之前的研究中，发现Cas9具有基因组规模的慢病毒单向导RNA文库，可以应用于功能丧失基因筛查，尽管功能丧失基因尚未在体外得到验证，而且这种方法尚未在猪细胞中使用。基于这些观察，慢病毒Cas9被用来感染猪原代成纤维细胞以获得携带Cas9核酸内切酶的细胞集落。随后，通过体细胞核移植产生表达四环素诱导的Cas9基因的猪胎儿成纤维细胞，并获得3个30天转基因猪胎儿成纤维细胞（PFF）。聚合酶链反应 (PCR)、逆转录 PCR 和蛋白质印迹分析表明 PFF 呈 Cas9 阳性。此外，三个整合之一位于 PFF1 细胞系中已知功能基因附近，而两个整合都不位于 PFF1 或 PFF2 细胞系中。据推测，这些转基因 PFF 可用于猪的条件基因组编辑，并可用于生成理想的改良猪模型。
Cas9 endonuclease, from so-called clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems of Streptococcus pyogenes, type II functions as an RNA-guided endonuclease and edits the genomes of prokaryotic and eukaryotic organisms, including deletion and insertion by DNA double‑stranded break repair mechanisms. In previous studies, it was observed that Cas9, with a genome‑scale lentiviral single‑guide RNA library, could be applied to a loss‑of‑function genetic screen, although the loss‑of‑function genes have yet to be verified in vitro and this approach has not been used in porcine cells. Based on these observations, lentiviral Cas9 was used to infect porcine primary fibroblasts to achieve cell colonies carrying Cas9 endonuclease. Subsequently, porcine fetal fibroblasts expressing the tetracycline‑inducible Cas9 gene were generated by somatic cell nuclear transfer, and three 30 day transgenic porcine fetal fibroblasts (PFFs) were obtained. Polymerase chain reaction (PCR), reverse transcription‑PCR and western blot analysis indicated that the PFFs were Cas9‑positive. In addition, one of the three integrations was located near to known functional genes in the PFF1 cell line, whereas neither of the integrations was located in the PFF1 or PFF2 cell lines. It was hypothesized that these transgenic PFFs may be useful for conditional genomic editing in pigs, and for generating ideal modified porcine models.